Reduced asymmetric dimethylarginine accumulation through inhibition of the type I protein arginine methyltransferases promotes renal fibrosis in obstructed kidneys
Ming Wu,*,1,2 Pinglan Lin,*,1 Lin Li,†,1 Dongping Chen,* Xuejun Yang,* Lin Xu,* Bing Zhou,‡ Chen Wang,‡ Yuanyuan Zhang,‡ Cheng Luo,‡ and Chaoyang Ye*,3
ABSTRACT:
The role of asymmetric dimethylarginine (ADMA) in chronic kidney disease (CKD) is unclear. Through inhibitionoftypeIproteinargininemethyltransferases(PRMTs),anovelstrategy,weaimedtodeterminetheeffect ofADMAonrenalfibrosisandexploreitsunderlyingworkingmechanisms.Aftershamorunilateralureterligation (UUO)operation,20–25gmalec57miceweretreatedwithvehicleorPT1001B,aninhibitoroftypeIPRMTs,for13d. Moreover, human kidney 2 (HK2) and normal rat kidney 49F (NRK-49F) cells were treated with various concentrationsofPT1001B orADMAinthepresenceof2.5ng/mlTGF-b.Wefoundthattreatmentwith PT1001Bincreased the deposition of extracellular matrix proteins, the expression of a smooth muscle actin, and connective tissue growthfactorinUUO-inducedfibrotickidneys,whichiscorrelatedwithreducedexpressionofPRMT1,reducedthe production of ADMA, and increased expression of uromodulin. In TGF-b–stimulated HK2 and NRK-49F cells, PT1001B dose-dependently inhibited ADMA production, increased NO concentrations, and enhanced the expression of profibrotic proteins. Exogenous addition of ADMA inhibited the expression of profibrotic proteins dose-dependently and attenuated the profibrotic effect of PT1001B. Moreover, ADMA reduced the NO concentration in PT1001B-treated HK2 cells. Finally, we conclude that ADMA has an antifibrotic effect in obstructed kidneys, and future application of type I PRMT inhibitor should be done cautiously for patients with CKD.—Wu, M., Lin, P., Li, L., Chen, D., Yang, X., Xu, L., Zhou, B., Wang, C., Zhang, Y., Luo, C., Ye, C. Reduced asymmetric dimethylarginineaccumulationthroughinhibitionofthetypeIproteinargininemethyltransferasespromotesrenal fibrosis in obstructed kidneys. FASEB J. 33, 000–000 (2019). www.fasebj.org
KEY WORDS: PRMT1 • ADMA • NO • CKD
Introduction
Chronic kidney disease (CKD) is defined based on the presence of renal damages and decrease in renal function (1). It affects 10% of adult population, and the prevalence of CKD is still increasing because of population aging, diabetes, hypertension, and other causes (1, 2). Tubulointerstitial fibrosis is a major hallmark of CKD, which is drivenbyrenaltubuleinjuries(3).Aftersevereorrecurrent injuries, renal tubular epithelial cells undergo changes with production of profibrotic cytokines, which initiate fibrotic responses (4).
Asymmetric dimethylarginine (ADMA) is a potent endogenous inhibitor of NOS, which can be produced by type I protein arginine methyltransferases (PRMTs) or inactivated by dimethylarginine dimethylaminohydrolase (DDAH) (5, 6). Circulating ADMA is a strong predictor for the progression of CKD (7, 8). Injection of recombinant adenovirus vector encoding DDAH-1 in CKD rats decreased plasma levels of ADMA, inhibited renal fibrosis, andpreventedthedeteriorationofrenalfunction(9).Global deletionofDdah1geneusingthegeneticapproachinmouse CKD models exacerbated kidney damages (10). Moreover, long-term administration of ADMA in CKD mice promoted renal fibrosis (11). However, a recent study showed that increased renal ADMA accumulation by kidneyspecific deletion of Ddah1 gene inhibited renal fibrosis and protectedrenalfunctionin2mousemodelsofCKD(6).This beneficial effect of ADMA in CKD was associated with reduced NO synthesis within the proximal tubule, which possiblyinducedtheup-regulationofuromodulin(UMOD) in the downstream thick ascending limb.
Type I PRMTs consist of 4 enzymes, among which PRMT1 is the predominant type I methyltransferase accounting for ;90% of all cellular arginine methylation events (5). PT1001B (formerly named Compound 28d, DCPR049_12) is a novel selective inhibitor of type I PRMTs, and it effectively displayed inhibition on proliferation of cancer cells, which was correlated with reduced cellular asymmetric arginine dimethylation levels, includinghistoneH4arginine3asymmetricdimethylation (H4R3me2a) (5).
Through inhibition of type I PRMTs, a novel strategy, we aimed to assess the effect of ADMA on renal fibrosis and explore its downstream working mechanism.
MATERIALS AND METHODS
Animal studies
Male c57 mice (specific pathogen-free grade) weighted between 20 and 25 g were purchased from the Shanghai Laboratory Animal Center (Shanghai, China). Animals were housed in the animal center of Shanghai University of Traditional Chinese Medicine according to local regulations and guidelines.
For unilateral ureter ligation (UUO) surgery, mice were anesthetized withsodiumpentobarbital(8mg/kg,i.p.),andthen a left flank incision was made to exposethe left ureter,which was ligated with 3-0 silk sutures. The same operation was performed in sham mice except for the ligation of ureter. A total of 29 mice were randomly divided into 4 groups: 1) sham + vehicle (saline) group (n = 7), 2) UUO + vehicle group (n = 7), 3) sham + PT1001B [30 mg/kg, synthesized by Wang et al. (5)] group (n = 7), and 4) UUO + PT1001B (30 mg/kg) group (n = 8). One day after the surgery, mice were treated with saline or 30 mg/kg PT1001B daily by intraperitoneal injection for 13 d. Kidney tissues were collected after euthanization at 14 d after the operation.
Animal experiments described herein were endorsed by the Animal Experimentation Ethics Committee of Shanghai University of Traditional Chinese Medicine (PZSHUTCM18111601).
Cell culture
Human kidney 2 (HK2) cells, a human renal proximal tubular epithelial cell line, were obtained from the Cell Bank of Shanghai Institute of Biologic Sciences (Chinese Academy of Science). Normal rat kidney 49F (NRK-49F) cells, a rat kidney interstitial fibroblast cell line, were purchased from National Infrastructure of Cell Line Resource, Chinese Academy of of Medical Sciences. HK2 and NRK-49F cells were cultured in DMEM/F12 medium containing 10% fetal bovine serum and 0.5% penicillin and streptomycin in an atmosphere of 5% CO2 and 95% air at 37°C. For Western blotting, HK2 and NRK-49F cells were seeded in 6-well plates to 40–50% confluence, and they were starved with DMEM/F12 medium with 0.5% fetal bovine serum overnight before the experiment. The next day, fresh medium containing 0.5% fetal bovine serum was changed, and then cells were exposed to 2.5 ng/ml TGF-b (PeproTech, Rocky Hill, NJ, USA) for 24 or 48 h in the presence of PT1001B, ADMA (APExBIO Technology, Houston, TX, USA), or a combination. For the NO measurement,HK2cellswereseededin 10-mmdishesto60–70% confluence and were starved with DMEM/F12 medium and 0.5% fetal bovine serum overnight before the experiment. The next day, fresh medium containing 0.5% fetal bovine serum was changed, and then cells were exposed to 2.5 ng/ml TGF-b for 4 h in the presence of PT1001B, ADMA, or a combination.
Measurement of NO
HK2 cells were lysed with Cell and Tissue Lysis Buffer for NO Assay (AU5800; Beyotime Biotech, Nantong, China) and centrifuged at 10,000 g at 4°C for 5 min. One hundred microliters of supernatant was collected. The intracellular NO content was assessed with Total NO Assay Kit (NO-180311-01; Fuyuan Biotech, Fuzhou, China) by Automatic biochemical analyzer (AU680;BeckmanCoulter,Brea,CA,USA)inClinicalLaboratory of Shuguang hospital. In brief, 20 ml of supernatant was mixed with 150 ml of 3-hydroxytyramine hydrochloride (12 mM) and incubated for 90 s at 37°C. Fifty microliters of 3-methyl-2benzothiazolinonehydrazone hydrochloride (20 mM) and phosphoric acid (10 mM) were added into the assay mixture and were followed by 30 s incubation at 37°C. The diversification of absorbance in 1.5 min was measured at 545 nm using a microplate reader. The content of NO was corrected with the protein concentration of each sample.
Masson’s trichrome and immunohistochemical staining
Kidneys were fixed in 4% paraformaldehyde and embedded in paraffin. Four-micrometer–thick sections of paraffin-embedded kidney tissue were subjected to immunohistochemical staining with anti-PRMT1 (1:100, 2449s; Cell Signaling Tehnology, Danvers, MA, USA) antibodies as previously described in Jing et al. For Masson staining, the 4-mm–thick sections of paraffinembedded kidney tissue were stained with hematoxylin and then with ponceau red liquid dye acid complex, which was followed by incubation with phosphomolybdic acid solution. Finally, the tissue was stained with aniline blue liquid and acetic acid. Images were obtained with the use of a microscope (Nikon 80i, Nikon, Tokyo, Japan). The Masson trichrome–positive tubulointerstitialarearelativetothewholeareafrom4randomcortical fields (magnification 3400) for each sample was analyzed using Imagine Pro software (Media Cybernetics, Rockville, MD, USA) and graphed.
Western blotting analysis
Protein was extracted from cells or mouse kidneys using RIPA lysis buffer (Beyotime Biotech). The protein concentration was measured by the Bradford method and then dissolved in Laemmli sample buffer. Samples were subjected to SDS-PAGE gels. After electrophoresis, proteins were electrotransferred to a PVDF membrane (Merck, Darmstadt, Germany), which was incubated in the blocking buffer (5% nonfat milk, 20 mM TrisHCl, 150 mM NaCl, pH 8.0, 0.1% Tween 20) for 1 h at room temperature and was followed by incubation with antibodies for fibronectin(FN;1:1000,ab23750;Abcam,Cambridge,MA,USA), connective tissue growth factor (CTGF; 1:1000, sc-373936 from Santa Cruz Biotechnology, Dallas, TX, USA, or ER1802-69 from HuaBio, Hangzhou, Zhejiang, China), collagen-I (Col-I; 1:1000, AF7001; Affinity, Cincinnati, OH, USA), a smooth muscle actin (a-SMA; 1:1000, GB13044 from Servicebio or ET1607-53 from HuaBio), glyceraldehyde-3-phosphate dehydrogenase (1:1000, 60004-1-lg; Proteintech, Wuhan, Hubei, China), PRMT1 (1:1000, 2449s; Cell Signaling Tehnology), ADMA (1:1000, 13522; Cell Signaling Tehnology), UMOD (1:1000, DF6692; Affinity), and a-tubulin (1:1000, AF0001; Beyotime Biotech) overnight at 4°C. BindingoftheprimaryantibodywasdetectedbyanECLmethod (BeyoECL Star, P0018A; Beyotime Biotech) using horseradish peroxidase–conjugated secondary antibodies (goat anti-rabbit IgG, 1:1000, A0208; Beyotime Biotech; or goat anti-mouse IgG, 1:1000, A0216; Beyotime Biotech). The band densities were measured using Quantity One software (Bio-Rad, Hercules, CA, USA).
Statistical analysis
Results were presented as means 6SD. Differences among multiple groups were analyzed by 1-way ANOVA, followed by a Newman-Keuls posthoc test. A value of P , 0.05 was considered statistically significant.
RESULTS
Inhibition of type I PRMTs aggravated renal fibrosis in UUO mice
Renal fibrosis was induced in 20–25 g wild-type c57 mice by UUO operation, which is the classic model to study renal fibrosis (13–15). One day after sham or UUO operations, mice were treated with vehicle or PT1001B, an inhibitor of type I PRMTs, for 13 d. Treatment with PT1001B had no effect on body weight of sham and UUO mice, and all mice survived during the treatment (unpublished results). Mild interstitial fibrosis was observed in vehicle-treated UUO mice compared with that in vehicle-treated sham mice (Fig. 1A). Treatment with PT1001B aggravated the development of renal interstitial fibrosis in UUO mice but not in sham mice (Fig. 1A).
The protein expression of FN, Col-I, CTGF, and a-SMA in mouse kidneys were assessed by Western blotting. As shown in Fig. 1B, the expression of FN, Col-I, CTGF, and a-SMA were significantly increased in UUO mouse kidneys compared with that in sham-operated mouse kidneys, and the treatment with PT1001B further enhanced the expression of these profibrotic proteins in UUO mouse kidneys (Fig. 1B).
Treatment with PT1001B inhibited the production of ADMA in UUO mouse kidneys
To determine the efficacy of PT1001B invivo, the expression of PRMT1, the H4R3me2a mark, and the production of ADMA were analyzed by Western blotting. As shown in Fig. 2A, the expression of PRMT1, H4R3me2a, and the production of ADMA were significantly increased in UUO mouse kidneys compared with that in sham-operated mouse kidneys, and PT1001B reduced the expression of PRMT1,H4R3me2a,andtheproductionofADMAinUUO mouse kidneys.
To further explore the mechanism of action of PT1001B, the expression of UMOD in mouse kidneys was measured. Figure 2B shows that UMOD expression in mouse kidneys was significantly up-regulated after UUO operation, and treatment with PT1001B further enhanced its expression in UUO kidneys, implying that NO production in UUO kidneys was possibly enhanced by PT1001B. Figure 2C shows that the NO concentration in whole mouse kidneys was significantly lower in the UUO group compared with that in the sham group, whereas treatment with PT1001B did not increase the concentration of NO in mouse kidneys in UUO group.
PT1001B increased NO content, inhibited ADMA production, and aggravated renal fibrosis in vitro
Immunohistochemistry staining analysis shows that PRMT1 is nuclear stained and strongly expressed in UUO kidneys compared with sham kidneys (Fig. 3A). The expression of PRMT1 is mainly localized in epithelial cells of renal tubules and can also be observed in interstitial cells (Fig. 3A). Figure 3B shows that 4 h of treatment with 2.5 ng/ml TGF-b did not change the NO concentration in HK2 cells. However, the concentration of NO was increased by PT1001B dosedependently at 1.67 and 15 mM in HK2 cells (Fig. 3B). In parallel, the expression of PRMT1 was significantly increased with stimulation of TGF-b for 4 h, which was inhibited by PT1001B dose-dependently in HK2 cells (Fig. 3B).
The direct effect of PT1001B on renal fibrosis was further studied in vitro. Figure 3C shows that 48 h of treatment with 2.5 ng/ml TGF-b significantly increased the expression of FN, CTGF, and a-SMA in HK2 cells, which were further enhanced by PT1001B in a dose-dependent manner at 1.67, 5, and 15 mM. The production of ADMA by HK2 cells was assessed by Western blotting. Figure 3C shows that after stimulation with TGF-b for 48 h, the production of ADMA was not increased, whereas it was inhibited by PT1001B dose-dependently in HK2 cells (Fig. 3C).
The direct effect of PT1001B on renal fibrosis was also assessed using renal fibroblasts. Figure 3D shows that 24 h of treatment with 2.5 ng/ml TGF-b significantly increased the expression of FN, CTGF, and a-SMA in rat renal interstitial fibroblasts NRK-49F, which were further enhanced by PT1001B in a dosedependent manner at 1.67 and 5 mM. NRK-49F cells were not tolerated with a high concentration of PT1001B, and dead cells were observed at 24 h after the treatment with 15 mM PT1001B. The production of ADMA by NRK-49F cells was also assessed by Western blotting. Figure 3D shows that after stimulation with TGF-b for 24 h, the production of ADMA was not increased, whereas it was inhibited by PT1001B dose-dependently in NRK-49F cells (Fig. 3D).
ADMA inhibited renal fibrosis in vitro
The effect of ADMA on NO synthesis was assessed. Treatmentwithdifferentconcentrations(10,30,100mM)of ADMA for 4 h did not change the content of NO in TGFb–stimulated HK2 cells (Fig. 4A). Treatment with ADMA at 100 mM for 24 or 48 h also did not change the NO concentrationinTGF-b–stimulatedHK2cells(unpublished results). Interestingly, 48 h of treatment with ADMA dose-dependently inhibited the expression of FN, CTGF, and a-SMA in TGF-b–stimulated HK2 cells (Fig. 4B). Moreover, the direct effect of ADMA on renal fibrosis wasalsoassessedusingrenalfibroblasts.Figure4Cshows that 24 h of treatment with ADMA dose-dependently inhibitedtheexpressionofFN,CTGF,anda-SMAinTGFb–stimulated NRK-49F cells.
ADMA mediated the effect of PT1001B on NO synthesis and renal fibrosis
Next, we investigated whether ADMA mediated the profibrotic effect of PT1001B. Figure 5A shows that 4 h of treatment with 15 mM PT1001B significantly increased the concentration of NO in TGF-b–stimulated HK2 cells, which was significantly reduced by the addition of 100 mM ADMA. The expression of PRMT1 was significantly reduced after 4 h treatment with 15 mM PT1001B in HK2 cells with or without the addition of 100 mM ADMA (Fig. 5A). Moreover, 48 h of treatment with PT1001B significantly increased the expression of FN, CTGF, and a-SMA in TGF-b–stimulated HK2 cells, and the addition of ADMA loweredtheexpression of FN,CTGF,and a-SMA in PT1001B-treated HK2 cells (Fig. 5B). In addition, the production of ADMA was significantly reduced by 48 h of treatment with PT1001B in TGFb–stimulated HK2 cells in the presence or absence of exogenous ADMA (Fig. 5B).
DISCUSSION
The role of ADMA in CKD is uncertain. It has long been regarded as a risk factor for CKD (8, 11). Continuous infusion of ADMA for 8 wk in the uninephrectomized mice induces hypertension, increases renal oxidative stress, and produces an important degree of glomerular and vascular fibrosis (11). However, a recent study shows that tubular-specific deletion of the Ddah1 gene increased renal ADMA and attenuated renal fibrosis in 2 animal models, suggesting that local ADMA rather than circulating ADMA inhibits renal fibrosis (6). In the current study, we employed a novel strategy to study the effect of ADMA on renal fibrosis. Our study shows that ADMA is an antifibrotic factor for CKD caused by ureter obstruction. First, we showed that PT1001B, a specific type I PRMT inhibitor, reduced renal ADMA production and aggravated renal fibrosis in a mouse model of UUO. Second, PT1001B dosedependently reduced ADMA production and enhanced the expression of profibrotic proteins in TGF-b–stimulated renal epithelial cells and renal fibroblasts. Third, the addition of ADMA dosedependently inhibited the expression of profibrotic proteins and blocked PT1001B-induced fibrotic responses in vitro.
The working mechanism of renal ADMA in CKD was thought to be related to the communication of NO synthesis within the proximal tubule with the expression of UMOD in the downstream thick ascending limb (6). Thus, we hypothesized that inhibition of ADMA by PT1001B promoted renal fibrosis by enhancing renal NO synthesis. However, we were not able to detect a significant increase in NO concentration in the whole UUO kidneys after PT1001B treatment, and we thought that the effect of PT1001B on NO synthesis is local and maybe restricted to renal tubules. Indeed, in the study by Tomlinson et al. (6), a significant change of NO concentration in renal tubular isolates of Ddah1PT2/2 mice, did not lead to the change of NO in whole kidney tissues. Interestingly, in the current study, we observed that PT1001B increased UMOD expression in UUO kidneys, implying that a local increase in NO synthesis within the proximal tubule may happen. To further support our hypothesis, we used TGF-b–stimulated HK2 cells as an in vitro model. We found that PT1001B dosedependently increased NO concentration in vitro, which was blocked by the addition of ADMA.
Surprisingly, we observed a direct antifibrotic effect of ADMA on renal epithelial cells. In TGF-b– stimulated HK2 cells, treatment with PT1001B enhanced the expression of profibrotic proteins, which was correlated with the reduced production of ADMA. In a further experiment, exogeneous ADMA dose-dependently reduced the expression of profibrotic proteins in HK2 cells. Moreover, addition of exogeneous ADMA blocked the profibrotic effect of PT1001B on renal epithelial cells.
With immunohistochemistry staining, we found that PRMT1 is localized not only in renal tubules but also in the interstitial areas in UUO kidneys, implying that PRMT1/ADMA modulates renal fibrosis also via renal fibroblasts. Indeed, through in vitro experiments, we observed the antifibrotic effect of ADMA in renal fibroblasts by using PT1001B or ADMA directly.
Arginine methylation is involved in many biologic processes through the mechanism of posttranslation modification of histones or nonhistone proteins (16, 17). For example, PRMT1 promotes hyperglycemia by methylating the transcriptional factor forkhead box O1 and thus increasing the nuclear retention and the transcriptional activity of transcriptional factor forkhead box O1 (17). In the current study, we found that the expression of PRMT1 and H4R3me2a are increased in fibrotic kidneys and reduced by the treatment with PT1001B, suggesting that PRMT1 may modulate renal fibrosis through histone modification– mediated epigenetic gene regulation or even nonhistone modification–mediated transcription factor activation, which go far beyond ADMA synthesis. Thus, the role of PRMT1 in renal fibrosis warrants further investigation.
There are several pathologic causes for renal fibrosis, including glomerular injuries, ischemia, toxic exposure, and obstruction (13, 18). The data from our study and from the previous study by Tomlinson et al. (6) only support that ADMA is antifibrotic in CKD, which is caused by ureter obstruction or toxic exposure. To address this limitation, we studied the effect of PT1001B or ADMA in vitro with renal tubular epithelial cells and renal fibroblasts, which reached the same conclusion.
In summary, activation of type I PRMTs in obstructed kidneys induces the accumulation of renal ADMA, which protects against renal fibrosis via a dual mechanism (Fig. 5C). On the one hand, the accumulation of renal ADMA results in reduced synthesis of NO within the proximal tubule, leading to the downregulation of UMOD expression in the downstream thick ascending limb and thus protects against renal fibrosis. On the other hand, ADMA directly inhibits the fibrotic changes of renal epithelial cells and renal fibroblasts in CKD.
Finally, we conclude that ADMA has an antifibrotic effect in obstructed kidneys, and future application of type I PRMT inhibitor should be done cautiously for patients with CKD.
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